One aspect of this project continues to be the generation of maximally efficient expression vectors for the specific antigens. We have generated a set of optimized expression vectors for HIV and SIV. HIV vectors are developed for eventual human clinical trials. In parallel, SIV expression vectors are developed and studied in the most faithful model system for human AIDS, ie., infection of Rhesus macaques by SIV, a virus closely related to HIV, which causes very similar pathology to human AIDS. Our results show that optimized DNA expression vectors in the absence of any other form of vaccine boosting are able to protect rhesus macaque from high viremia after challenge with a highly pathogenic SIVmac251 challenge. We have developed sets of plasmids expressing the majority of HIV or SIV antigens. These plasmids have entered human clinical trials through our CRADA collaborators. They have also been used in several collaborative vaccine studies and were shown to provide excellent priming in DNA-prime-virus boost vaccinations. We have also proposed to use DNA vaccination for periodically boosting the immune response. This comes from the realization that DNA vaccination boosts existing immune responses with every application and does not have the problem of viral vectors, which focus immune response to the vaccine vector. To further improve vaccine efficiency we study the intrinsic properties of the different candidate antigens. We have shown that modulating the form, stability and cellular fate of the DNA produced antigens has profound effects on their immunogenicity and the type of response generated. We perform comparative studies to develop optimal forms of several antigens. In addition to antigen modification, we study the role of molecular adjuvants such as cytokines, chemokines and costimulatory molecules for the improvement of DNA vaccines. We have shown great increases in expression and function of optimized cytokine vectors for IL-12 and IL-15. IL-12 used as a DNA vaccine adjuvant increased dramatically the cellular and humoral immune response of macaques to SIV. We presently test whether optimized use of IL-15 may also improve the longevity and quality of immune response. The optimal use of these powerful cytokines is of great interest also in the cancer immunotherapy field. We have used our optimized DNA vectors in therapeutic vaccination of macaques chronically infected with SIVmac251. We showed an increased immune response after DNA vaccination of infected macaques and a strong decrease of viral load compared to unvaccinated controls. These results support the development of DNA vaccination for therapeutic immunization of infected persons. Our experiments have highlighted the necessity for adequate antigen expression for a successful vaccination. We have developed rational methods to quantify expression and to correlate antigen levels and immunogenicity in an effort to further optimize vaccine outcome. We have shown that in vivo electroporation greatly induces cellular and humoral immune responses after DNA vaccination. We optimize this new delivery method in primates for eventual human use.